Much focus has shifted towards understanding how glial dysfunction contributes to age-related neurodegeneration due to the critical roles glial cells play in maintaining healthy brain function. Cell-cell interactions, which are largely mediated by cell-surface proteins, control many critical aspects of development and physiology; as such, dysregulation of glial cell-surface proteins in particular is hypothesized to play an important role in age-related neurodegeneration. However, it remains technically difficult to profile glial cell-surface proteins in intact brains. Here, we applied a cell-surface proteomic profiling method to glial cells from intact brains in Drosophila, which enabled us to fully profile cell-surface proteomes in-situ, preserving native cell-cell interactions that would otherwise be omitted using traditional proteomics methods. Applying this platform to young and old flies, we investigated how glial cell-surface proteomes change during aging. We identified candidate genes predicted to be involved in brain aging, including several associated with neural development and synapse wiring molecules not previously thought to be particularly active in glia. Through a functional genetic screen, we identified one surface protein, DIP-β, which is down-regulated in old flies and can increase fly lifespan when overexpressed in adult glial cells. We further performed whole-head single-nucleus RNA-seq, and revealed that DIP-β overexpression mainly impacts glial and fat cells. We also found that glial DIP-β overexpression was associated with improved cell-cell communication, which may contribute to the observed lifespan extension. Our study is the first to apply in-situ cell-surface proteomics to glial cells in Drosophila, and to identify DIP-β as a potential glial regulator of brain aging.

Endocytosis actively remodels the neuronal surface proteome to drive diverse cellular processes, yet its global extent and effects on neural circuit development have defied comprehensive interrogation. Here, we introduce endocytome profiling: a systematic, cell-type-specific approach for mapping cell-surface protein (CSP) dynamics in situ. Quantitative proteomic analysis of developing Drosophila olfactory receptor neuron (ORN) axons generated an endocytic atlas comprising over 1,000 proteins and revealed the extent to which the cell-surface proteome is remodeled to meet developmental demands. Targeted interrogation of a junctional CSP showed that its endosome-to-surface ratio is precisely balanced to enable developmental axon pruning while preserving mature axon integrity. Multi-omic integration uncovered widespread transcellular signaling and identified a growth factor secreted by neighboring neurons to direct ORN axon targeting via endocytic regulation of its receptor. Endocytome profiling provides unprecedented access to cell-surface proteome dynamics and offers a platform to dissect proteome-scale remodeling across diverse cell types and contexts.

Tumors can exert a far-reaching influence on the body, triggering systemic responses that contribute to debilitating conditions like cancer cachexia. To characterize the mechanisms underlying tumor-host interactions, we utilized a BioID-based proximity labeling method to identify proteins secreted by Ykiact adult Drosophila gut tumors into the bloodstream/hemolymph. Among the major proteins identified are coagulation and immune-responsive factors that contribute to the systemic wasting phenotypes associated with Ykiact tumors. The effect of innate immunity factors is mediated by NFκB transcription factors Relish, dorsal, and Dif, which in turn upregulate the expression of the cachectic factors Pvf1, Impl2, and Upd3. In addition, Ykiact tumors secrete Eiger, a TNF-alpha homolog, which activates the JNK signaling pathway in neighboring non-tumor cells, leading to cell death. The release of damage-associated molecular patterns (DAMPs) from these dying cells presumably amplifies the inflammatory response, exacerbating systemic wasting. Targeting the inflammatory response, the JNK pathway, or the production of cachectic factors could potentially alleviate the debilitating effects of cancer cachexia.

Proximity labeling coupled with mass spectrometry is a powerful method for studying living systems with high spatial and temporal resolution. This perspective aims to provide a history of how APEX- and TurboID-based proximity labeling technologies were developed through dedication, innovation, and, most importantly, collaboration. We share our candid guidance on how to execute a successful proximity labeling experiment and comments on future developments in this field.

Introduction Multiple Myeloma (MM) precursors Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM) have variable risk of progression to MM and identifying which patients may progress is challenging. Bone marrow (BM) biopsies are used for staging and identifying high-risk events associated with progression. However, they are invasive and cannot be repeated often for monitoring tumor burden. Proteome profiling of peripheral blood (PB) plasma may advance non-invasive precursor disease staging, monitoring and characterization. Here, we performed comprehensive plasma proteomic profiling across the MM disease continuum, including progressive and stable disease, to provide biological insights and identify protein-based markers of high-risk disease for improved prognostication. Methods We executed high-throughput plasma proteomic profiling for ~3000 proteins using the Olink® Explore 3072 library and Proximity Extension Assay (PEA) technology. We profiled 462 PB plasma samples from 351 individuals, including MGUS (n=66), SMM (n=174), MM (n=49), and healthy donors (n=98). Samples from patients with progressive disease (n=32) and stable disease (n=32) with matched clinical follow-up time were also profiled; 17/32 patients with progressive disease had sequential samples from both precursor and active disease, while 15/32 patients had a precursor stage sample only. Precursor PB samples ranged 1.04-6.91 years (median of 2.33 years) prior to MM progression. T-tests, ANOVAs, and a linear mixed effect model were used to identify significant proteins across disease stages and progression status. Results were adjusted for multiple testing using the Benjamini-Hochberg Method. A subset of individuals also underwent single-cell RNA sequencing (scRNA-seq) of tumor and immune cells from paired PB/BM from the same proteomics timepoint to enable cellular mapping of signals detected in the plasma. Results We captured high levels of plasma cell surface proteins, including BCMA, SLAMF7, CD38 and FCRL5, highlighting the utility of PEA technology to monitor soluble levels of clinically relevant targets. We analyzed functional protein networks showing stepwise dysregulation over disease progression and identified enrichment of proteins involved in immune evasion, cell motility, inflammation and cell adhesion. Correlation analysis of proteins with clinical features demonstrated BCMA and TACI levels had a strong combined positive correlation with BM plasma cell infiltration, M-protein and FLC ratio. Additional proteins, FCRL5, CD79B, MZB1, CD48, FCRLB, LY9 and QPCT, showed moderate positive correlations specifically with BM infiltration, suggesting their potential as surrogate markers for BM tumor burden and/or for improving risk prediction in routine blood-based assessments of precursor patients. We next aimed to improve the discrimination of disease states by training a machine learning-based classifier using plasma proteomic features and assigning samples to disease stages. We demonstrated 97% SMM/MM samples could be identified from healthy samples, indicating our classifier could confidently screen disease-related cases. Moreover, 85% of SMM samples were correctly classified as SMM, while misclassified cases labelled as MM exhibited early signs of progression, suggesting that the plasma proteome may provide earlier indications of evolving disease. Next, by evaluating protein levels in patients with progressive and stable SMM disease, we identified a prognostic five-protein signature that was significantly elevated at the precursor stage timepoint of patients who progressed to active MM. Validation of the signature in an external international cohort collected from three institutions confirmed that 4 of the 5 proteins were indeed significantly elevated in patients with progressive disease. Finally, integrative analysis of scRNA-seq of tumor/immune cells and plasma proteomics was used to elucidate cell-type level information of the signature proteins. Four of the proteins were predominately expressed in malignant vs. non-malignant plasma cells and/or memory B-cells, suggesting the signature partially provides a readout of malignant plasma cell biology. Conclusion Overall, we characterized dysregulated protein networks across disease stages, developed a plasma-based classifier for accurate stage classification, and identified and validated a prognostic protein signature associated with progressive disease.

Reactivation of fetal hemoglobin (HbF) expression has been a target for the treatment of hemoglobinopathies such as sickle cell disease (SCD) and β-thalassemia for several decades. While gene therapy has recently been approved as a cure for these hemoglobinopathies, its high cost limits global implementation. Hydroxyurea thus remains the treatment of choice for the induction of HbF although there is variability in the degree of response in compliant patients. Significant efforts have thus been aimed at identifying additional potent inducers of HbF. In vitro studies have documented immunomodulatory drugs (IMiDs), such as pomalidomide, as potent pharmacological inducers of HbF. The primary mechanism of action of IMiDs is the induction of proteasomal degradation of intracellular proteins mediated through Cereblon-dependent ubiquitination. Specific genes in multiple myeloma and del5q-syndrome have been identified as IMiDs' targets; however, the upstream mediators of pomalidomide leading to HbF induction remain to be defined. Previous studies identified IKZF1, IKZF3, CSK1α and ZFP91 as direct IMiD-mediated targets of Cereblon. In a previous study, we ruled out a potential role for IKZF1 and IKZF3 in HbF induction. To identify novel targets of pomalidomide involved in HbF regulation, we performed a global proteomic analysis on healthy human bone marrow CD34+ cells cultured with pomalidomide during ex vivo erythropoiesis. Among the 12,000 quantified proteins, we confirmed the downregulation of known pomalidomide targets such as IKZF1 and ZFP91. Interestingly, we identified FIZ1 (FLT3-Interacting Zinc Finger 1) as a novel target whose protein level was significantly reduced by pomalidomide and this effect was dose-dependent (starting at 100nM of pomalidomide). Having identified FIZ1 as a novel pomalidomide target, we sought to investigate its function in human erythropoiesis. CRISPR/Cas9 was used to knockout FIZ1 in CD34+ cells at day 2 of differentiation, and editing efficiency was assessed by measuring the indel frequency. At day 4 and day 11 of in vitro differentiation, there were no significant differences in dynamics erythropoiesis between control and sgFIZ1 knockout conditions, as measured by flow cytometry using CD105/CD71 (Day 4, progenitor stages) or Band3/a4-integrin (Day 11, precursor stages) as markers of erythroid differentiation. Western blot analyses demonstrated that the expression levels of key erythroid transcription factors (e.g. GATA1, KLF1) were also unaltered in FIZ1 knockout cells. Having ruled out that FIZ1 knockout had any detrimental effects on adult human erythropoiesis, we assessed its impact on HbF production. At day 14 of culture, we observed a significant increase in the total expression levels of γ-globin, while β-globin was slightly decreased and α-globin was unchanged. Similarly, quantification of F-cells by flow cytometry showed a significant increase in the F-cell population in FIZ1 knockout cells (51% vs 6% in the control). Mechanistically, FIZ1 knockout led to decreased expression of HbF repressors BCL11A, HRI and SOX6 while expression of other HbF regulators such as LRF remained unchanged at day 7 of differentiation. Similar expression patterns were noted in cells treated with pomalidomide and an additive effect was observed when cells were treated with both FIZ1 guides and pomalidomide. In summary, our data indicate that pomalidomide enhances the degradation of FIZ1 leading to increase in γ-globin expression without altering erythroid differentiation and as such is a novel inducer of HbF is erythroid cells.

Kidney cancer frequently causes paraneoplastic syndromes, including hypercalcemia and cachexia, but the underlying mechanisms are incompletely understood. The most common form of kidney cancer, clear cell renal cell carcinoma, is frequently caused by loss of the pVHL tumor suppressor protein and the resulting upregulation of the HIF2 transcription factor. We show that PTHLH, which resides on a ccRCC amplicon on chromosome 12p, is a direct HIF2 transcriptional target in ccRCC. Further, we show that the increased PTHLH expression is both necessary and sufficient for the induction of hypercalcemia and cachexia in preclinical orthotopic cell line tumor models. Consistent with these observations, two different allosteric HIF2 inhibitors, belzutifan and NKT2152, rapidly ameliorated hypercalcemia and cachexia in ccRCC patients, including in some patients who did not exhibit objective tumor shrinkage.

Enzymes that oxidize aromatic substrates have been harnessed for cell-based technologies including proximity labeling and electron microscopy; however, they are associated with drawbacks such as the need for toxic H2O2. Here, we explore multicopper oxidases (laccases) as a new enzyme class for proximity labeling and electron microscopy in mammalian cells. LaccID was generated through 11 rounds of directed evolution from an ancestral fungal laccase and catalyzes one-electron oxidation of diverse aromatic substrates using O2 instead of toxic H2O2. Surprisingly, we found that LaccID is selectively active at the surface plasma membrane of both living and fixed cells. We use LaccID proximity labeling and mass spectrometry to map the changing surface proteome of T cells that engage with tumor cells through antigen-specific T cell receptors. In addition, we use LaccID as a genetically encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID.

The DNA-incorporating nucleoside analogs azacytidine (AZA) and decitabine (DEC) have clinical efficacy in blood cancers, yet the precise mechanism by which these agents kill cancer cells has remained unresolved - specifically, whether their anti-tumor activity arises from conventional DNA damage or DNA hypomethylation via DNA methyltransferase 1 (DNMT1) inhibition. This incomplete mechanistic understanding has limited their broader therapeutic application, particularly in solid tumors, where early clinical trials showed limited efficacy. Here, through the assessment of drug sensitivity in over 600 human cancer models and comparison to a non-DNA-damaging DNMT1 inhibitor (GSK-3685032), we establish DNA hypomethylation, rather than DNA damage, as the primary killing mechanism of AZA and DEC across diverse cancer types. In further support of an epigenetic killing mechanism, CRISPR drug modifier screens identified a core set of chromatin regulators, most notably the histone deubiquitinase USP48, as AZA and DEC protective factors. We show that USP48 is recruited to newly hypomethylated CpG islands and deubiquitinates non-canonical histones, establishing USP48 as a key molecular link between the two components of epigenetic gene regulation: DNA methylation and chromatin modification. Furthermore, loss of USP48, which occurs naturally through biallelic deletions in human cancers, sensitized both hematologic and solid tumors to DNMT1 inhibition in vitro and in vivo. Our findings elucidate the epigenetic mechanism of action of AZA and DEC and identify a homeostatic link between DNA methylation and chromatin state, revealing new therapeutic opportunities for DNMT1 inhibitors in solid tumors.

UM171 is a potent agonist of ex vivo human haematopoietic stem cell self-renewal1. By co-opting KBTBD4, a substrate receptor of the CUL3-RING E3 ubiquitin ligase (CRL3) complex, UM171 promotes the degradation of the LSD1-CoREST corepressor complex, thereby limiting haematopoietic stem cell attrition2,3. However, the direct target and mechanism of action of UM171 remain unclear. Here we show that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1/2 to promote corepressor degradation. Through proteomics and chemical inhibitor studies, we identify the principal target of UM171 as HDAC1/2. Cryo-electron microscopy analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex identifies an asymmetric assembly in which a single UM171 molecule enables a pair of KELCH-repeat propeller domains to recruit the HDAC1 catalytic domain. One KBTBD4 propeller partially masks the rim of the HDAC1 active site, which is exploited by UM171 to extend the E3-neosubstrate interface. The other propeller cooperatively strengthens HDAC1 binding through a distinct interface. The overall CoREST-HDAC1/2-KBTBD4 interaction is further buttressed by the endogenous cofactor inositol hexakisphosphate, which acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces is demonstrated by base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, we reveal how the cooperativity offered by a dimeric CRL3 E3 can be leveraged by a small molecule degrader.

Cell surface proteins (CSPs) regulate key cellular functions and represent valuable targets for diagnostics and therapeutics. Despite advances in proteomic workflows, CSP analysis from cryopreserved or low-input clinical samples remains limited by technical constraints, including reduced membrane integrity, inefficient labeling, and high background. To address these challenges, we optimized and benchmarked two complementary surface enrichment strategies compatible with low-input applications (fewer than 1 million cells) and real-world sample types, including fresh, viably cryopreserved, and dissociated solid tissues. We systematically compared oxidation-based N-glycoprotein capture and WGA-HRP-mediated proximity labeling across a range of input amounts using both solid tumor (A549) and hematologic cancer (KMS-12-BM) cell lines. The N-glycopeptide method yielded superior specificity in low-input contexts, while WGA-HRP captured complementary CSP subsets. Together, the methods identified more than 700 CSPs, with approximately 175 unique identifications per protocol. Both workflows detected dynamic EGFR internalization following EGF stimulation and maintained high reproducibility (Pearson correlation greater than 0.9) between fresh and cryopreserved preparations. To extend these findings to tissue-derived samples, we optimized dissociation protocols for healthy endometrium and applied the N-glycopeptide method to cryopreserved dissociated endometrium from three healthy donors. Enzymatic dissociation enabled accurate CSP profiling from fewer than 1 to 2 million cells. This study provides a systematic comparison of two leading surface proteomics approaches, validates their performance on cryopreserved and low-input specimens, and demonstrates applicability to clinically relevant tissues. Our optimized workflows enable robust surfaceome characterization in translational settings where sample quantity and preservation methods are often limiting, opening new avenues for biomarker discovery and patient stratification.

Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H2O2. Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O2 instead of toxic H2O2, and exhibits activity selective to the surface plasma membrane of both living and fixed cells. We show that LaccID can be used with mass spectrometry-based proteomics to map the changing surface composition of T cells that engage with tumor cells via antigen-specific T cell receptors. In addition, we use LaccID as a genetically-encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID.

Dendritic cells (DCs) are specialized sentinel and APCs coordinating innate and adaptive immunity. Through proteins on their cell surface, DCs sense changes in the environment, internalize pathogens, present processed Ags, and communicate with other immune cells. By combining chemical labeling and quantitative mass spectrometry, we systematically profiled and compared the cell-surface proteomes of human primary conventional DCs (cDCs) in their resting and activated states. TLR activation by a lipopeptide globally reshaped the cell-surface proteome of cDCs, with >100 proteins upregulated or downregulated. By simultaneously elevating positive regulators and reducing inhibitory signals across multiple protein families, the remodeling creates a cell-surface milieu promoting immune responses. Still, cDCs maintain the stimulatory-to-inhibitory balance by leveraging a distinct set of inhibitory molecules. This analysis thus uncovers the molecular complexity and plasticity of the cDC cell surface and provides a roadmap for understanding cDC activation and signaling.

IRE1α is an endoplasmic reticulum (ER) sensor that recognizes misfolded proteins to induce the unfolded protein response (UPR). We studied cholera toxin (CTx), which invades the ER and activates IRE1α in host cells, to understand how unfolded proteins are recognized. Proximity labeling colocalized the enzymatic and metastable A1 segment of CTx (CTxA1) with IRE1α in live cells, where we also found that CTx-induced IRE1α activation enhanced toxicity. In vitro, CTxA1 bound the IRE1α lumenal domain (IRE1αLD), but global unfolding was not required. Rather, the IRE1αLD recognized a seven-residue motif within an edge β-strand of CTxA1 that must locally unfold for binding. Binding mapped to a pocket on IRE1αLD normally occupied by a segment of the IRE1α C-terminal flexible loop implicated in IRE1α oligomerization. Mutation of the CTxA1 recognition motif blocked CTx-induced IRE1α activation in live cells, thus linking the binding event with IRE1α signal transduction and induction of the UPR.

O-GlcNAcylation was identified in the 1980s by Torres and Hart and modifies thousands of cellular proteins, yet the regulatory role of O-GlcNAc is still poorly understood compared to the abundance of mechanistic information known for other cycling post-translational modifications like phosphorylation. Many challenges are associated with studying O-GlcNAcylation and are tied to the technical hurdles with analysis by mass spectrometry. Over the years, many research groups have developed important methods to study O-GlcNAcylation revealing its role in the cell, and this perspective aims to review the challenges and innovations around O-GlcNAc research and chronicle the work by Donald F. Hunt and his laboratory, particularly in development of ETD and its application to this field of research.

The recent technological and computational advances in mass spectrometry-based single-cell proteomics have pushed the boundaries of sensitivity and throughput. However, reproducible quantification of thousands of proteins within a single cell remains challenging. To address some of those limitations, we present a dedicated sample preparation chip, the proteoCHIP EVO 96 that directly interfaces with the Evosep One. This, in combination with the Bruker timsTOF demonstrates double the identifications without manual sample handling and the newest generation timsTOF Ultra identifies up to 4000 with an average of 3500 protein groups per single HEK-293T without a carrier or match-between runs. Our workflow spans 4 orders of magnitude, identifies over 50 E3 ubiquitin-protein ligases, and profiles key regulatory proteins upon small molecule stimulation. This study demonstrates that the proteoCHIP EVO 96-based sample preparation with the timsTOF Ultra provides sufficient proteome depth to study complex biology beyond cell-type classifications.

Mass spectrometry (MS)-based single-cell proteomics (SCP) has gained massive attention as a viable complement to other single cell approaches. The rapid technological and computational advances in the field have pushed the boundaries of sensitivity and throughput. However, reproducible quantification of thousands of proteins within a single cell at reasonable proteome depth to characterize biological phenomena remains a challenge. To address some of those limitations we present a combination of fully automated single cell sample preparation utilizing a dedicated chip within the picolitre dispensing robot, the cellenONE. The proteoCHIP EVO 96 can be directly interfaced with the Evosep One chromatographic system for in-line desalting and highly reproducible separation with a throughput of 80 samples per day. This, in combination with the Bruker timsTOF MS instruments, demonstrates double the identifications without manual sample handling. Moreover, relative to standard high-performance liquid chromatography, the Evosep One separation provides further 2-fold improvement in protein identifications. The implementation of the newest generation timsTOF Ultra with our proteoCHIP EVO 96-based sample preparation workflow reproducibly identifies up to 4,000 proteins per single HEK-293T without a carrier or match-between runs. Our current SCP depth spans over 4 orders of magnitude and identifies over 50 biologically relevant ubiquitin ligases. We complement our highly reproducible single-cell proteomics workflow to profile hundreds of lipopolysaccharide (LPS)-perturbed THP-1 cells and identified key regulatory proteins involved in interleukin and interferon signaling. This study demonstrates that the proteoCHIP EVO 96-based SCP sample preparation with the timsTOF Ultra provides sufficient proteome depth to study complex biology beyond cell-type classifications.

In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.